Monoclonal antibody of antiplague bacillus F1 antigen and application of monoclonal antibody

A monoclonal antibody, F1 antigen technology, applied in applications, antibodies, antibacterial drugs, etc., can solve the problems of poor affinity and specificity of monoclonal antibodies, no mention of protective effect, low immunogenicity, etc., and achieve high specific binding activity. Effect

Active Publication Date: 2015-01-21
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0004] In 2010, Xiao et al. reported that one anti-F1 Fab and two anti-LcrV Fabs were screened from a non-immune human phage library. When they were converted into full-molecule human antibodies, the F1 monoclonal antibody could protect 1 out of 6 mice from survival. , while the two anti-LcrV monoclonal antibodies have no protective effect alone, and the three monoclonal antibodies can protect the survival of 5 out of 6 mice (Xiaodong Xiao et al, PLOS ONE, 2010, 5(10): 1-12 .)
The advantage of this method is that the screened monoclonal antibody sequence is fully human, and the immunogenicity in vivo is low. The disadvantage is that the monoclonal antibody obtained has poor affinity and specificity without primary immunization and booster immunization, and the protective effect is difficult to obtain. Mutual corroboration with the results reported in the literature
In 2005, Yin Jiaxiang et al. reported to obtain 3 strains of monoclonal antibodies against F1 antigen, but their protective effects were not mentioned (Yin Jiaxiang et al., Chinese Journal of Endemic Diseases: 2005, 24(5))
As far as the existing technology is concerned, there is still a lack of plague antibodies that not only have the advantages of low immunogenicity in humans, but also exhibit high binding activity and good protective effect on the target antigen F1

Method used

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  • Monoclonal antibody of antiplague bacillus F1 antigen and application of monoclonal antibody
  • Monoclonal antibody of antiplague bacillus F1 antigen and application of monoclonal antibody
  • Monoclonal antibody of antiplague bacillus F1 antigen and application of monoclonal antibody

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Embodiment 1

[0029] The preparation of embodiment 1 chimeric antibody

[0030] 1. Cloning of mouse monoclonal antibody gene

[0031] 1.1 Preparation of mouse monoclonal antibody

[0032] Using recombinant plague capsular antigen F1 as the immunogen, a high-affinity and high-specificity mouse-derived anti-F1 monoclonal antibody F2H5 hybridoma cell line was obtained by conventional monoclonal antibody preparation methods, and the subtype of the secreted antibody molecule is IgG1 Subclass, the light chain is the Kappa subtype.

[0033]1.2 Cloning of the variable region gene of the monoclonal antibody F2H5 light and heavy chain

[0034] The monoclonal antibody is composed of a variable region and a constant region. Because the gene variation of the variable region of the mouse monoclonal antibody is large, and the gene sequence of the constant region is very conservative, the 5'-RACE method is used for cloning. A primer sequence was added to the 5' end, and PCR amplification was performed w...

Embodiment 2

[0095] Example 2 Preparation of mouse-derived monoclonal antibody from ascites and analysis of its binding activity with antigen

[0096] 7-10 days before the inoculation of the tumor strain, each mouse was intraperitoneally injected with 0.5 ml of paraffin oil, and the abdomen of the mouse was gently rubbed with a cotton ball to fully diffuse the paraffin oil in the mouse abdominal cavity. Prepare hybridoma cells, recover and observe their growth status, select those cultures with good cell shape and activity, when they are in the logarithmic growth phase, gently blow down with RPMI l640, centrifuge and remove bovine serum in the culture medium , suspended in saline, and injected into the peritoneal cavity of human mice. The amount of cells injected into each mouse l0 5 –l0 6 . 8-10 days after the inoculation of the tumor strain, there may be accumulation of ascites. When the abdomen is obviously swollen, the ascites should be collected. Centrifuge 4mL of ascitic fluid at...

Embodiment 3

[0097] Embodiment 3.ELISA detects the binding activity of chimeric antibody and F1

[0098] Dilute the recombinant F1 to 2μg / ml, coat the enzyme-linked plate with 100ul / well, overnight at 4°C, block with blocking solution (20mM PB, 0.15M NaCl, 5% milk powder) the next day at 37°C for 1h, wash the plate, and add serial dilutions Wash the plate after incubating at 37°C for 1 hour, add 1:1 000 diluted HRP-labeled goat anti-human Fc (Sigma, A0170) and incubate at 37°C for 1 hour, wash the plate, develop color with OPD, and measure OD with a microplate reader 450 . See Figure 9 . Serially diluted monoclonal antibodies 256, 128, 64, 32, 16, 8, 4, 2ng / ml react with coated recombinant F1, showing a dose-effect response, with a sensitivity of 4-8ng / ml, and recombinant anthrax protective antigen PA, Recombinant plague V antigen, recombinant tetanus antigen, etc. did not react (not shown in the figure). It shows that the monoclonal antibody against plague F1 antigen provided by the ...

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Abstract

The invention discloses a monoclonal antibody of an antiplague bacillus F1 antigen and an application of the antibody in preparing a plague treatment drug. The antibody disclosed by the invention has specific antigen associativity, low immunogenicity and a good animal protective function.

Description

technical field [0001] The present invention relates to the gene of anti-plague monoclonal antibody and the polypeptide encoded by the gene, as well as the application of the gene and polypeptide in the preparation of novel therapeutic medicine for plague. Background technique [0002] Plague is a natural foci disease that can spread independently in animals. There are various types of animal natural foci in the world, and it is difficult to completely eliminate them. Human plague still occurs in 11 provinces and regions with epidemic foci in my country. In the autumn of 2010, human plague broke out in Tibet, my country. In addition, Yersinia pestis has been listed as an important biological warfare agent and bioterrorist agent and has been highly valued. More importantly, the discovery of multidrug-resistant Yersinia pestis made the treatment of plague even worse. The "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" stipulates t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/85C12N5/10A61K39/40A61P31/04
CPCY02A50/30
Inventor 陈薇李建民张金龙任军李冰宋小红于婷刘树玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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